1. Field of the Invention
This invention relates generally to the field of therapeutic agents that have anti-mycobacterial activity. More particularly, this invention relates to halogenated compounds that have anti-Mycobacterium tuberculosis activity, therapeutic agents for treating tuberculosis and methods of treating tuberculosis.
2. Description of Related Art
Tuberculosis is the oldest documented infectious disease, and it remains an important global health problem. An estimated 1 billion people worldwide are infected with Mycobacterium tuberculosis; 8 to 10 million new tuberculosis cases occur each year, and the number of new cases is estimated to increase to 12 million in the year 2005. Inadequacy of diagnosis and prevention in addition to inefficient treatment programs account for uncontrolled infection in developing countries.
Therapies exist to treat tuberculosis, however tuberculosis is not entirely cured by present drug treatments. Current drugs can minimize relapse rates with optimal treatment. With the best available chemotherapy, tubercle bacilli are slowly disposed of or killed. The widespread use of some drugs, such as isoniazid, has resulted in the development of resistant strains such that current drugs fail to eradicate some Mycobacterial infections. Therefore new drugs with anti-mycobacterial action are essential to successfully treat tuberculosis infections.
Because Mycobacteria develop resistance to drugs, optimal anti-tuberculous therapies require the use of several drugs in combination. Mycobacterial populations contain spontaneous mutants that are resistant to drugs even prior to exposure. The frequency of such mutations can vary between 1 in 30 less than 100 to 1 in greater than 10,000, depending upon the drug. Single drug therapy can inhibit the majority of organisms in an infected site, yet permit, and in fact encourage, uncontrolled growth of the resistant mutants. Early combination therapy with at least two drugs is the preferred method of preventing emergence of large resistant populations in the original tuberculous cavities. Some drugs are most valuable for their ability to suppress emergence of resistance during combination therapy. An example is p-aminosalicylic acid, which can delay development of streptomycin resistance.
Thus, anti-mycobacterial agents can be important not only for their own efficacy against susceptible organisms but for their ability to enhance effectiveness of other agents by controlling emergence of resistant populations, for example populations resistant to pyrazinamide. Pyrazinamide is a major drug used in the therapy of tuberculosis and the synthesis of pyrazinamide was described by Kushner et al, J. Am. Chem. Soc. 74:3617 (1952), and the compound was patented in 1954 as a tuberculostatic agent (U.S. Pat. No. 2,677,641 issued to Williams). When pyrazinamide is used alone resistance develops quickly, and for this reason it is usually administered in combination with other drugs such as isoniazid. Pyrazinamide is also hepatotoxic, which further limits its use as a therapeutic agent.
The development of new anti-mycobacterial agents presents a challenge of balancing toxicity to mycobateria with patient safety. Due to fluorine""s unique chemistry, fluorinated compounds offer some desirable features in pharmacological applications. For example, fluorine is the second smallest element, after hydrogen, and thus, fluorine closely mimics hydrogen at enzyme receptor sites. Fluorine""s high electronegativity typically alters chemical reactivity at these enzyme sites, and enzyme deactivation can result. However, high electronegativity also increases oxidative and thermal stability as a Cxe2x80x94F bond is stronger than a Cxe2x80x94H bond, which can also affect enzymatic activity. In some cases (e.g., 5-fluorouracil), the specific location of a xe2x80x9cdeceptorxe2x80x9d fluorine instead of hydrogen blocks, an essential biochemical reaction. The presence of fluorine may also promote lipid solubility, thereby enhancing drug absorption and transport rates in vivo.
Fluorinated organic molecules can be effective in the treatment of a variety of disorders. However, fluorination of compounds for the treatment of M. tuberculosis has not previously been successful. Isoniazid is one of the most active drugs for the treatment of tuberculosis. Fluorination of the pyridine ring of isoniazid resulted in drastically decreasing activity against M. tuberculosis. 
The global resurgence of tuberculosis and development of drug resistant populations have rekindled the need for and interest in the development of new anti-tubercular drugs. However no new anti-tuberculosis agents have been developed since the introduction of rifampin into clinical use. There continues to be a need for new compounds with high efficacy in anti-tuberculosis activity for use as therapeutic agents.
These needs are met by the halogenated compounds of this invention, which possess high anti-tuberculosis activity or are useful as intermediates in the manufacture of such compounds.
In one embodiment of this invention, a class of compounds which possess high anti-tuberculosis activity includes:
a halogenated compound having Structure I or a pharmaceutically acceptable salt thereof: 
wherein X1 is a halogen and X2 is a second halogen or hydrogen, and Y is sulfur or oxygen; and,
a halogenated compound having Structure II: 
or a pharmaceutically acceptable salt thereof.
In another embodiment of this invention, a class of compounds which possess high anti-tuberculosis activity includes:
a halogenated compound having Structure IV or a pharmaceutically acceptable salt thereof: 
wherein X1 is a halogen and X2 is a second halogen or hydrogen;
a halogenated compound having Structure V or pharmaceutically acceptable salt thereof: 
wherein X is a halogen; and
a halogenated compound having Structure VI: 
or a pharmaceutically acceptable salt thereof.
A further embodiment of this invention, is a composition, which possess high anti-tuberculosis activity comprising any one of the halogenated compounds of this invention and a pharmaceutically acceptable binder, wherein the halogenated compound has anti-mycobacterium activity.
A still further embodiment of this invention is a method of treating a mammal infected with a Mycobacterium, comprising administering to the mammal a non-toxic, effective amount of a composition comprising any one of the halogenated compounds of this invention and a pharmaceutically acceptable binder, wherein the halogenated compound has anti-mycobacterium activity.
A still further embodiment of this invention is a halogenated compound having Structure III: 
wherein the compound of Structure III is useful as an intermediate in the manufacture of compounds of Structure II.
The novel halogenated compounds of this invention which are halogenated derivatives of two synthetic anti-tuberculosis agents, thioacetazone and p-aminosalicylic acid, have been synthesized. Halogenation (noted by X1 or X2) may be at any unsubstituted ring position in the structure. In general, the halogenated compound of this invention has the structure of Structure I: 
wherein X1 is a halogen and X2 is a second halogen or hydrogen, and Y is sulfur or oxygen; or,
has the structure of Structure IV: 
wherein X1 is a halogen and X2 is a second halogen or hydrogen. Alternatively, compounds of this invention may be pharmaceutically acceptable salts of compounds having Structures I and/or IV. Typical pharmaceutically acceptable salts include hydrochloride salts, hydrobromide salts, sulfate salts, and the like. The halogenated derivatives of Structures I and IV possess anti-mycobacterial activity and are particularly useful for the treatment of tuberculosis. In particular, fluorinated, chlorinated, brominated and iodinated analogs of thioacetazone and fluorinated analogs of p-amino-salicylic acid have been synthesized for use as anti-tuberculosis therapeutic agents either alone or in combination with other conventional anti-tuberculosis theraputic agents.
During the screening of intermediates from the synthesis of sulfathiadiazoles, benzaldehyde thiosemicarbazone was shown to be active against tuberculosis. Structural modification produced the 4-acetamido derivative, thioacetazone. 
The mechanism of action is not known. Studies have shown that the thiosemicarbazones are not competitive inhibitors of p-aminobenzoic acid, and there is no cross-resistance with isoniazid.
Replacement of the thiosemicarbazone group with a semicarbazone, hydrazone, or oxime yields inactive compounds. Substitution on the primary amines of the thiosemicarbazone group with one or two alkyl groups or the sulfur atom with oxygen or nitrogen results in loss of activity. The order of activity of p-substitutions is:
(CH3)2CHNH greater than NH2xe2x95x90CH3CONHxe2x95x90(CH3)2N greater than NO2
The fluoro derivative of thioacetazone was synthesized using the following reactions. In the following synthesis schemes and examples major reactants and products are identified with a bold face number; and the acronyms ACN, Ac, and Et have their conventional meaning, i.e., respectively acrylonitrile, acetic, and ethyl. 4-Acetamido-3-fluorobenzaldehyde 15 was synthesized from 4-acetamidobenzaldehyde 14 through a reaction with Selectfluor(trademark) fluorinating agent (Aldrich #43,947-9, [1-(chloromethyl)-4-fluoro-1,4-diazoniabicyclo[2.2.2]octane bis(tetrafluoro-borate)]). 
The product was characterized as 4-acetamido-3-fluorobenzaldehyde 15. 4-Acetamido-3-fluorobenzaldehyde 15 reacts with thiosemicarbazide to yield 4-acetamido-3-fluorobenzaldehyde thiosemicarbazone 16. Compound 16 has been tested and shown to be both non-toxic and highly active against M. Tuberculosis. 
Synthesis of a positional isomer of compound 16, 4-acetamido-2-fluorobenzaldehyde thiosemicarbazone 17, and other halogenated analogs of thiacetazone are also described herein, as halogenated compounds of the present invention.
The 2-fluoro positional isomer may be synthesized through several approaches. In one approach, 4-acetamido-2-fluorobenzaldehyde thiosemicarbazone 17 is prepared using the following approach.
4-Cyano-3-fluoroacetanilide 21 is first prepared in the following reaction scheme: 
in which 3-fluoroacetanilide 22 is converted to 4-cyano-3-fluoroacetanilide 21. 4-Acetamido-2-fluorobenzaldehyde 20 is next synthesized by reducing the nitrile derivative, 4-cyano-3-fluoroacetanilide 21, with Raney nickel, as illustrated below. 
4-Acetamido-2-fluorobenzaldehyde 20 then is reacted with thiosemicarbazide to form 4-acetamido-2-fluorobenzaldehyde thiosemicarbazone 17 in 45% yield. An exemplary scheme for this reaction is shown below. 
Chlorination of 4-acetamidobenzaldehyde 14 using NaOCl as a chlorinating reagent results in the chloro derivative 4-acetamido-3-chlorobenzaldehyde 24, as illustrated below. 
The reaction of 4acetamido-3-chlorobenzaldehyde 24 with thiosemicarbazide, shown below, forms thiosemicarbazone 25 in 90% yield. 
Bromination of 4-acetamidobenzaldehyde 14 with Br2/AcQH results in a solid mixture of three compounds, as detected by GC-MS (gas chromatograph-mass spectrometer). The three compounds are 4-acetamido-3-bromobenzaldehyde 26, 4-bromoacetanilide 27, and 2,4-dibromoacetanilide 28, as shown below. 
Compound 26, 4-acetamido-3-bromobenzaldehyde, contains a formyl group, and reacts with thiosemicarbazide to produce 4-acetamido-3-bromobenzaldehyde thiosemicarbazone 29 as shown below. 
Iodination of 4-aminobenzonitrile 30 with ICI produces 4-amino-3-iodobenzonitrile 31. Acetylatation of 4-amino-3-iodobenzonitrile 31 results in compound 32, which can be reduced with Raney nickel to form 4-acetamido-3-iodobenzaldehyde 33. Reaction of compound 33 with thiosemicarbazide yields 4-acetamido-3-iodobenzaldehyde thiosemicarbazone 34, as illustrated below. 
p-Aminosalicylic Acid (identified hereinafter as PAS) 7 is an anti tuberculosis agent, however PAS has little effect on the respiration of M. tuberculosis. PAS is only effective against growing bacilli and the anti-tuberculosis activity of PAS is reversed with p-aminobenzoic acid. These indications suggest that PAS has a mechanism of action similar to that of sulfonamides.
In previous attempts, PAS has not been successfully modified into an anti-tuberculosis agent. Unless the PAS molecule is readily regenerated, modification to the structure of PAS typically results in loss of activity. Such modifications include: 1) primary amino group replacement with hydroxy, alkoxy, tertiary amines, or amides; 2) masking the hydroxyl group as an ether or ester; 3) replacing the hydroxyl group with a thiol or an amino group; 4) converting the carboxylic acid group to alkyl esters, amidines, amides, or nitrates.
Methyl 4-acetamidosalicylate 10, may be synthesized from PAS. This protected form of PAS, may be formed via esterification of the carboxylic acid group, followed by acetylation of the amine group, as shown below. 
Methyl 4-acetamidosalicylate 10 may be reacted with 1.5 equimoles of Selectfluor(trademark), yielding a product characterized as 4-acetamido-5-fluorosalicylic acid methyl ester 11 as illustrated below. 
Hydrolysis of methyl 4-acetamido-5-fluorosalicylate 11 in 10% sodium hydroxide yields compound 12, as shown below. 
Testing and analysis of the halogenated compounds of the present invention were conducted using standard practices administered through the TAACF (Tuberculosis Antimicrobial Acquisition and Coordinating Facillity). The program is coordinated under the direction of the U.S. National Institute of Allergy and Infectious Diseases (NIAID), Southern Research Institute.
The pharmaceutical composition of this invention comprises a halogenated compound and a pharmaceutically acceptable binder, wherein the halogenated compound is the halogenated thioacetazone previously described, the halogenated p-aminosalicylic acid previously described; or a combination thereof. The halogenated compound of this composition is an active ingredient in the composition having anti-mycobacterium activity, and may be used with one or more other conventional anti-mycobacterium agents such as isoniazid, rifampin, ethambutol and streptomycin. As used herein the term xe2x80x9cpharmaceutically acceptable binderxe2x80x9d is intended to have the conventional meaning of a non-toxic inert substance combined with the active ingredient for preparing an agreeable or convenient dosage form (i.e., an excipient). The pharmaceutical compositions containing the halogenated compound of this invention, is characterized by being active against at least one of the following Mycobacteria: Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis Erdman, Mycobacterium avium (American Type Culture Collection [ATCC] 25291), isoniazid-resistant Mycobacterium tuberculosis (ATCC 35822), rifampin-resistant Mycobacterium tuberculosis (ATCC 35838), ethambutol-resistant Mycobacterium tuberculosis, kanamycin-resistant Mycobacterium tuberculosis, ciprofloxacin-resistant Mycobacterium tuberculosis or a combination thereof.
The pharmaceutical compositions containing the halogenated compound of this invention, may be in a form suitable for oral use, for example as tablets, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparations. Various pharmaceutically acceptable binders or excipients useful in the present invention are disclosed in columns 4-6 of U.S. Pat. No. 4,962,111, the disclosure of which is incorporated herein by reference.
The pharmaceutical compositions of this invention are particularly useful for treating a mammal infected with a Mycobacterium, by administering to the mammal a non-toxic, effective amount of a composition comprising the halogenated thioacetazone, the halogenated p-aminosalicylic acid of this invention, or a combination thereof; and a pharmaceutically acceptable binder. The compositions are particularly useful in treating a mammal infected with Mycobacterium tuberculosis. 
Primary screening of anti-mycobacterial activity was conducted at 6.25 xcexcg/mL (or molar equivalent of highest molecular weight compound in a series of congeners) against Mycobacterium tuberculosis H37Rv (ATCC 27294) in BACTEC(trademark) 12B medium using a broth microdilution assay. Specifically, the Microplate Alamar Blue Assay (hereinafter xe2x80x9cMABAxe2x80x9d) was used. Compounds exhibiting fluorescence were tested in the BACTEC(trademark) 460 radiometric system.
Some of the compounds demonstrating at least 90% inhibition in the primary screen were retested at lower concentrations against M. tuberculosis H37Rv to determine the actual minimum inhibitory concentration (hereinafter xe2x80x9cMICxe2x80x9d) using MABA. The MIC is defined as the lowest concentration effecting a reduction in fluorescence of 90% relative to controls.
Concurrent with the determination of MICs, compounds were tested for cytotoxicity (1C50) in VERO cells at concentrationsxe2x89xa662.5 xcexcg/mL or 10xc3x97 the MIC for M. tuberculosis H37Rv (when solubility in media permitted). After 72 hours exposure, viability was assessed on the basis of cellular conversion of MTT into a formazan product using the Promega CellTiter 96 Non-radioactive Cell Proliferation Assay.
Compounds for which the selectivity index, SI (i.e., 1C50:MIC ratio), was greater than 10 had in vitro activity confirmed by the BACTEC(trademark) 460 radiometric system at 6.25 ug/mL. Compounds were then tested for killing of M. tuberculosis Erdman (ATCC 35801) in monolayers of mouse bone marrow macrophages. Compounds were tested at 4-fold concentrations equivalent to 0.25, 1, 4, and 16xc3x97 the MIC. The test measured EC90 and EC99 values, which are the lowest concentration effecting a 90% and 99% reduction, respectively, in colony forming units at seven days as compared to drug-free controls.
Concurrent with the testing of compounds in macrophages, MICs were determined in a MABA against a strain of M. avium (ATCC 25291) and against three strains of singly-drug-resistant (SDR) M. tuberculosis. Each SDR strain is resistant to a single anti-tuberculosis drug). Compounds were tested against M. tuberculosis strains resistant to isoniazid (ATTC 35822), rifampin (ATCC 35838), and one additional SDR strain chosen on the basis of compound type (thiacetazone-resistant M. tuberculosis in the case of structure I and PAS-resistant M. tuberculosis in the case of structure IV). Confirmatory testing also occurred against drug-sensitive M. tuberculosis strains H37Rv and Erdman. The minimum bactericidal concentration (MBC) was then determined for M. tuberculosis H37Rv and Erdman (and for the appropriate drug-resistant strain, for analogs of known anti-tubercular drugs) by subculturing onto drug-free solid media and enumerating colony forming units following exposure in supplemented Middlebrook 7H9 media to drug concentrations equivalent to and higher than the previously determined MICs of the respective strains.
Compounds were tested for their capacity to inhibit the growth of virulent M. tuberculosis in a realistic in vivo aerosol mouse model. Mice were exposed to an aerosol of M. tuberculosis Erdman, which deposits approximately 50 bacilli into the lungs of the animal. The course of the infection is then followed in the lungs and spleen for 50 days by plating homogenates of harvested organs [n=5] on nutrient agar and determining bacterial numbers. As the growing infection was slowly controlled and contained, a peak number of about log 5.0 was observed in the infected lungs.
Test compounds were administered to groups of mice starting on day 20 post-inoculation. Three dose levels of drug were given (generally intraperitoneal) once per day, or oral gavage twice per day); an additional group was given isoniazid as a positive control. Bacterial numbers were assessed on days 35 and 50, and compared to untreated control values. The data are expressed as the log10 protection provided by a given dose of the compound against the growth of the organism in the untreated control group. Statistical tests are also applied to the raw data to determine levels of significance. For new compound classes it may be necessary to determine a minimum toxic dose (MTD) before running the animal model.
Higher level evaluation of compounds against M. avium is available for compounds showing an M. avium MICxe2x89xa66.25 xcexcg/mL. Expanded primary screening was conducted at a range of 1 xcexcg/mL-64 xcexcg/mL against five M. avium clinical isolates (strains 100, 101, 108, 109, 116) in Middlebrook 7H9 broth using a MABA and a BACTEC 460 system.
Compounds with MICxe2x89xa68 xcexcg/mL in at least three of the five strains tested were retested at lower concentrations against 30 strains, including five strains resistant to clanthromycin (MIC  greater than 32 xcexcg/mL). Compounds that demonstrated significant activity against the panel of 30 strains were tested against three M. avium strains (100, 101, 109) representing the three serotypes encountered in AIDS patients (8, 1, 4, respectively). This test measures intracellular activity of the compound in an infected macrophage model using the human monocyte cell line U937. Potential synergism with ethambutol is examined by adding ethambutol (4 xcexcg/mL) to the compound.
In vivo activity was studied in a mouse model for M. avium infection. Beige-C57BL/J bg female mice were infected I.V.(intravenous) with 3xc3x97107 cfu of bacilli. After one week, therapy was initiated and continued for four weeks. The liver and spleen were aseptically dissected, weighed, and homogenized. Serial dilutions of the liver and spleen tissues were plated onto 7H1 1 agar for quantitative culture.